Vibrational spectroscopic and microscopic imaging studies of biomineralization: From models to tissues

Bone mineralization is a very important biological process and a complicated one. Mineralized tissues are formed, matured and remodeled to maintain a system in dynamic equilibrium as well as to fulfill their bodily functions. Different spatial regions of bone have different structures. This heterogeneity of bone mineralization almost certainly has physiological consequences. Several diseases affect mineralized tissue. A better understanding of the properties and mechanisms of bone mineralization may shed light on those problems. Vibrational spectroscopy can provide direct information about molecular structures and interactions. To simplify the complexity of the system, in vitro models are used to help understand the spectra and to develop spectra-structure correlations for molecules under controlled conditions. A relatively new technology, Fourier Transform Infrared Imaging (FTIRI) is applied to obtain spatially resolved molecular structure information at ∼6--7 mum spatial resolution.;Amorphous calcium phosphate (ACP) in vitro is a precursor to the major inorganic part of the bone, hydroxyapatite (HA). As the ACP to HA transition takes place, the inorganic mineral changes from an amorphous phase to a well-crystallized phase and the spectra change accordingly. Raman spectroscopy was first used to follow the process. Changes have been observed in phosphate nu1 region. FTIR spectroscopic studies revealed that spectral features of both phosphate nu3 and nu4 regions change during the process. 2D-correlation analysis has been performed on both regions to reveal subtle changes in spectra. The general similarity of the correlation pattern of the model to the real tissue has also been demonstrated.;Carbonate ions can be readily substituted into the hydroxyapatite lattice. The crystal sizes of hydroxyapatite particles change with different levels of carbonate substitution. A series of type-B carbonated hydroxyapatites were synthesized and their infrared spectra taken. Spectra-structure correlations were established. The change in crystal sizes was monitored by X-ray powder diffraction and was consistent with previous studies. To quantitatively determine carbonate weight percentage in mineralized tissue, an IR spectral parameter was established to correlate with the amount of carbonate substitution. Combining this parameter with FTIR imaging, a detailed map of carbonate distribution was generated for the first time and it is consistent with chemical analysis.;Fourier Transform Infrared Imaging (FTIRI) is a powerful technology, which combines the structure information from mid-infrared spectra with the spatial information obtained from an IR microscope and array detector. A method to extend this approach to three dimensions has also been developed and 3D chemical images of several parameters have been generated.;The process of fracture healing was investigated in a rat model. Transverse fractures were created in rat femurs. Pieces of femur sections at the fracture site following sacrifice were studied by FTIRI. Infrared parameters of these sections were calculated to access the quality of the callus in the healing process. The results were compared with a parallel experiment in which rats received estrogen treatment following fracture.